Analysis of HSA-PAA Complexation Using SEC-SAXS Combination: Unraveling Stoichiometry, Reversibility, and Interaction Specificity.

Abstract

This work leverages the integration of size exclusion chromatography (SEC) with small-angle X-ray scattering (SAXS) to investigate the complex interactions between human serum albumin (HSA) and poly(acrylic acid) (PAA). The SEC-SAXS approach is proven in this study to effectively eliminate aggregates, enhancing data quality and revealing intricate details of protein-polymer associations. Initial findings demonstrate that HSA maintains its native structure across pH 5-8 and that HSA shows no significant interaction with the neutral polyethylene glycol (PEG). This highlights the critical role of charge regulation and electrostatic forces in HSA-PAA complex formation previously reported and the specificity of this interaction. The study further reveals that the HSA-PAA complex stoichiometry is highly dependent on PAA size, with larger PAA chains forming more elongated structures. The binding stoichiometry is then shown to increase nonlinearly, suggesting a delicate balance between attractive HSA-PAA and repulsive HSA-HSA interactions. Notably, HSA-PAA complexes exhibit reversible behavior, dissociating at pH > 5 and in media devoid of PAA.